The ubiquitously expressed adaptor protein Grb2 is crucial for the activation and propagation of signaling pathways that control many human physiological processes. Along with its role in propagating signaling pathways downstream of activated receptors and adaptor proteins, the disregulated formation of Grb2-mediated complexes has been linked to numerous pathological conditions in humans, including cancer, diabetes, autoimmune disorders, allergies/asthma and cardiovascular disease. Grb2 is composed of a Src homology 2 (SH2) domain, which binds specific phosphorylated tyrosines, flanked by two Src homology 3 (SH3) domains, which associate with proline- rich sequences. The interaction of Grb2 with SH2 and SH3 domain ligands has been extensively examined. Because of this, Grb2 is the primary model system for our understanding of the activation and function of SH and SH3 domain-containing proteins. Although these studies have provided insight into potential ligands and the binding mechanism of Grb2 and other important signaling proteins, they have not clearly addressed several critical questions. An examination of these questions is important if we are to fully understand how Grb2 and other related proteins regulate the formation and function of clinically relevant complexes. Our long-term goal is to characterize how SH2 and SH3 domain- containing proteins control the formation and function of clinically relevant signaling complexes in order to facilitate the develop of therapeutic regimens for diseases linked to the disregulated formation of these complexes. The objective of this specific proposal is to comprehensively examine the Grb2- mediated complexes that form at an SH2 domain ligand, the adaptor protein LAT. Our central working hypothesis is that Grb2 forms cooperative interactions with both LAT and individual SH3 domain ligands that control the formation and function of these critical multiprotein signaling complexes. To test this hypothesis, we will 1) molecularly characterize the interaction of Grb2 with the SH3 domain ligands Sos1, HPK1 and c-Cbl, 2) determine the forces that drive the association of specific Grb2 SH3 domain ligands with LAT and 3) determine the role of SH3 domain ligands in the interaction of Grb2 with LAT. To achieve these aims, we will employ several innovative, state-of-the-art biophysical, and biochemical techniques, including our new, more robust analysis methods for isothermal titration calorimetry and sedimentation velocity analytical ultracentrifugation. The information gained from these studies will allow us to significantly advance our understanding of the binding mechanism of Grb2 and other related proteins to both SH2 and SH3 domains ligands. This will not only provide new insight into the formation of SH2 and SH3 domain-mediated signaling complexes, but will also facilitate the production and/or use of novel therapeutic agents for the treatment of debilitating human diseases. PUBLIC HEALTH RELEVANCE: This proposal will examine how cooperative protein-protein interactions control the specificity and binding capacity of the SH2 and SH3 domains of the ubiquitously expressed adaptor protein Grb2. The knowledge gained from this study will help to us to understand the activation and function of Grb2 and other related clinically important proteins, which is expected to provide critical information for the rational design and/or use of inhibitors for these proteins. These agents will be useful for the treatment of multiple human diseases, including cancer, cardiovascular disease, autoimmune diseases, asthma/ allergies and the rejection of transplanted organs.